Method for synthesizing cDNA

ABSTRACT

A method for synthesizing cDNA characterized by performing a reverse transcription reaction in the presence of an enzyme having a reverse transcriptional activity and another enzyme different from the former one which as a 3′-5′ exonuclease activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/201,314, filed Jul. 24, 2002, which is a divisional of U.S.application Ser. No. 09/856,539, now issued as U.S. Pat. No. 6,485,917.U.S. application Ser. No. 09/856,539 is a 371 national stage applicationof PCT/JP99/06560 filed Nov. 25, 1999.

TECHNICAL FIELD

The present invention relates to a novel method for synthesizing a cDNAand a kit used for the method, which are useful in a field of geneticengineering.

BACKGROUND ART

Analysis of mRNA molecules derived from various genes is very importantin order to elucidate biological phenomena. Discovery of anRNA-dependent DNA polymerase, so-called a reverse transcriptase, from aretrovirus has enabled a reverse transcription reaction in which a cDNAis synthesized using an RNA as a template. As a result, methods foranalyzing mRNA molecules have made rapid progress. Then, the methods foranalyzing mRNA molecules using a reverse transcriptase have now becomeindispensable experimental methods for studying genes. Furthermore,these methods, which have been applied to cloning techniques and PCRtechniques, have become indispensable techniques not only for studyinggenes but also in wide variety of fields including biology, medicine andagriculture.

However, the conventional reverse transcription methods had manyproblems such as interruption of a cDNA synthesis reaction, inability tosynthesize a long cDNA, low fidelity, and damage of a template RNA inthe course of reaction due to long time required for the reaction.

It is considered that the interruption of the cDNA synthesis reaction isdue to the secondary structure formed by the RNA as a template. Optimalreaction temperature for a reverse transcriptase from a retrovirus islow. RNAs form complicated secondary structures during the reaction atthe temperature. Then, the cDNA synthesis reaction is interrupted at thesites of such secondary structures. A method in which a heat-resistantreverse transcriptase is used has been proposed in order to solve theabove-mentioned problem. However, the reactivity of this method is notsatisfactory. In addition, reverse transcriptases have not been known toexhibit a proofreading activity during a reverse transcription reaction,and a method for synthesizing a cDNA with high fidelity has not beenknown.

As described above, it was difficult to synthesize a cDNA with highefficiency and with high fidelity according to the conventional methods.Thus, a more efficient method for synthesizing a cDNA has been desired.

OBJECTS OF THE INVENTION

The present invention has been made in view of the prior art asdescribed above. The main object of the present invention is to solvethe problems associated with the prior art and to provide a method forsynthesizing a cDNA with high reaction efficiency and accuracy.

SUMMARY OF THE INVENTION

The present invention is outlined as follows. The first aspect of thepresent invention relates to a method for synthesizing a cDNA,characterized in that the method comprises conducting a reversetranscription reaction in the presence of an enzyme having a reversetranscription activity and another enzyme having a 3′-5′ exonucleaseactivity.

The second aspect of the present invention relates to a method foramplifying a cDNA, characterized in that the method comprises conductinga gene amplification reaction using a cDNA synthesized according to themethod of the first aspect as a template.

The third aspect of the present invention relates to a kit for cDNAsynthesis, which contains an enzyme having a reverse transcriptionactivity and another enzyme having a 3′-5′ exonuclease activity.

The fourth aspect of the present invention relates to a kit foramplifying a cDNA by conducting a gene amplification reaction using acDNA synthesized according to the method of the first aspect as atemplate, which contains an enzyme having a reverse transcriptionactivity and another enzyme having a 3′-5′ exonuclease activity as wellas a reagent for the gene amplification reaction.

The present inventors have found the efficiency of cDNA synthesis, andthe fidelity are increased by conducting a reverse transcriptionreaction in the presence of an enzyme having a 3′-5′ exonucleaseactivity in a cDNA synthesis reaction. Thus, the present invention hasbeen completed.

DETAILED DESCRIPTION OF THE INVENTION

One of the main features of the method for synthesizing a cDNA of thepresent invention is that a cDNA is synthesized using an RNA as atemplate in a reverse transcription reaction system containing an enzymehaving a 3′-5′ exonuclease activity.

Examples of the samples containing RNAs which can be used in the methodof the present invention include, but are not limited to, samples fromorganisms such as a cell, a tissue and a blood, and samples that maycontain an organism such as a food, a soil and a waste water. The samplemay be a preparation containing a nucleic acid obtained by processingthe above-mentioned sample according to a known method. Examples of thepreparations that can be used in the present invention include a celldestruction product or a sample obtained by fractionating the product,the total RNA in the sample, or a sample in which specific RNA moleculessuch as mRNAs are enriched.

The RNAs to which the method of the present invention can be appliedinclude, but are not limited to, RNA molecules such as total RNA, mRNA,tRNA and rRNA in a sample, as well as specific RNA molecules (e.g., RNAmolecules each having a common base sequence motif, transcripts obtainedusing an RNA polymerase and RNA molecules concentrated by a subtractionmethod). Any RNAs for which a primer used for a reverse transcriptionreaction can be prepared may be used.

The primer used for synthesizing a cDNA from an RNA as a template in thepresent invention is not limited to specific one as long as it is anoligonucleotide that has a nucleotide sequence complementary to that ofthe template RNA and that can anneal to the template RNA under reactionconditions used. The primer may be an oligonucleotide such as anoligo(dT) or an oligonucleotide having a random sequence (a randomprimer).

In view of specific annealing, the length of the primer is preferably 6nucleotides or more, more preferably 10 nucleotides or more. In view ofoligonucleotide synthesis, the length is preferably 100 nucleotides orless, more preferably 30 nucleotides or less. The oligonucleotide can besynthesized using, for example, the DNA synthesizer type 394 fromApplied Biosystems Inc. (ABI) according to a phosphoramidite method.Alternatively, any methods including a phosphate triester method, anH-phosphonate method and a thiophosphonate method may be used tosynthesize the oligonucleotide. The oligonucleotide may be derived froma biological sample. For example, it may be isolated and prepared from aDNA prepared from a natural sample digested with a restrictionendonuclease. In view of synthesis of a cDNA from a template RNA, theconcentration of the primer in the reverse transcription reactionmixture is preferably 0.1 μM or more, more preferably 0.5 μM or more. Inview of inhibition of the reaction, the concentration is preferably 10μM or less, more preferably 5 μM or less.

Any enzymes having reverse transcription activities can be used in thepresent invention as long as they have activities of synthesizing cDNAsusing RNAs as templates. However, enzymes having reverse transcriptionactivities at a high temperature (i.e., heat-resistant reversetranscriptases) are preferable for the purpose of the present invention.Examples of such enzymes which can be used include a DNA polymerase froma bacterium of genus Thermus (e.g., Tth DNA polymerase) and a DNApolymerase from a thermophilic bacterium of genus Bacillus. The presenceof a manganese ion in a reaction mixture is indispensable for theexertion of the reverse transcription activity of Tth DNA polymerase.The manganese ion is known to reduce the fidelity of a PCR. Thus, it isrequired to eliminate the manganese ion when a reverse transcriptionreaction mixture in which Tth DNA polymerase is used is used for a PCR.DNA polymerases from thermophilic bacteria of genus Bacillus do notrequire the addition of a manganese ion for the exertion of its reversetranscription activity and the removal thereof upon a PCR. In thisregard, DNA polymerases from thermophilic bacteria of genus Bacillus arepreferable for the present invention. A DNA polymerase from Bacilluscaldotenax (hereinafter referred to as Bca DNA polymerase) and a DNApolymerase from Bacillus stearothermophilus (hereinafter referred to asBst DNA polymerase) are preferable. These enzymes do not require amanganese ion for the reactions. Furthermore, they can be used tosynthesize a cDNA while suppressing the formation of secondary structureof the template RNA under high temperature conditions.

Bacillus caldotenax is a thermophilic bacterium having an optimal growthtemperature of about 70° C. Bca DNA polymerase from this bacterium isknown to have a DNA-dependent DNA polymerase activity, an RNA-dependentDNA polymerase activity (a reverse transcription activity), a 5′-3′exonuclease activity and a 3′-5′ exonuclease activity.

The enzyme may be either an enzyme purified from its original source ora recombinant protein produced by using genetic engineering techniques.The enzyme may be subjected to modification such as substitution,deletion, addition or insertion by genetic engineering techniques orother means. Examples of such modified enzymes include BcaBEST DNApolymerase (Takara Shuzo), which is Bca DNA polymerase lacking its 5′-3′exonuclease activity, and Bst DNA polymerase, Large fragment (NewEngland Biolabs), which is Bst DNA polymerase lacking its 5′-3′exonuclease activity. The above-mentioned enzymes lacking their 5′-3′exonuclease activities can be preferably used in the present inventionin particular.

The amount of the enzyme having a reverse transcription activity to beused is not specifically limited. The enzyme may be used, for example,in an amount used in a conventional reverse transcription reaction. Theamount of the enzyme having a reverse transcription activity may beincreased as compared with that for a conventional method to conduct thecDNA synthesis reaction more efficiently and to shorten the reactiontime. For example, when BcaBEST DNA polymerase is used for conducting areverse transcription reaction in a reaction volume of 20 μl, the amountof the enzyme in the reaction mixture is 0.5 U or more. In view of cDNAsynthesis efficiency, it is preferably 22 U or more, more preferably 42U or more. The activity of a DNA polymerase as described herein is basedon the indication for a commercially available enzyme. An activity ofincorporating 10 nmol of total nucleotides into an acid-insolubleprecipitate in 30 minutes under reaction conditions suitable for the DNApolymerase is defined as 1 U. A DNA as a template and a reactiontemperature suitable for each DNA polymerase are used. For example, inthe case of BcaBEST DNA polymerase, an activity of incorporating 10 nmolof total nucleotides into an acid-insoluble precipitate in 30 minutes at60° C. using polydeoxy (ATP-TTP) as a template/primer is defined as 1 U.

The method for synthesizing a cDNA of the present invention ischaracterized in that it comprises conducting a reverse transcriptionreaction in the presence of an enzyme having a 3′-5′ exonucleaseactivity. The enzyme having a 3′-5′ exonuclease activity to be used inthe present invention is not limited to specific one as long as it hasthe activity. For example, a DNA polymerase having a 3′-5′ exonucleasecan be used. Examples of such enzymes include α-type DNA polymerasessuch as a DNA polymerase from a bacterium of genus Pyrococcus (Pfu DNApolymerase (Stratagene), Pyrobest DNA polymerase (Takara Shuzo), DeepVent DNA polymerase (New England Biolabs), KOD DNA polymerase (Toyobo),Pwo DNA polymerase (Boehringer), etc.) and a DNA polymerase from abacterium of genus Thermococcus (Vent DNA polymerase (New EnglandBiolabs), etc.), and pol I-type DNA polymerases such as a DNA polymerasefrom Escherichia coli (polymerase I, Klenow fragment, etc.) and a DNApolymerase from a bacteriophage (T4 DNA polymerase, etc.). Preferably,an α-type DNA polymerase which exhibits a strong 3′-5′ exonucleaseactivity is used. The pol I-type DNA polymerase or the α-type DNApolymerase refers to a series of enzymes classified on the basis of theamino acid sequence homology. The features of the amino acid sequencesare described in Nucleic Acids Research, 15:4045-4657 (1991).

For the purpose of the present invention, an enzyme that acts on a3′-terminus of a DNA hybridized with an RNA is used. Furthermore, anenzyme that exhibits a 3′-5′ exonuclease activity at a high temperatureis preferable. In this regard, an α-type DNA polymerase derived from ahyperthermophilic archaebacterium is preferable. The α-type DNApolymerase derived from a hyperthermophilic archaebacterium isexemplified by a DNA polymerase from a bacterium of genus Pyrococcus.

A cDNA can be amplified by conducting a nucleic acid amplificationreaction using the cDNA obtained according to the method described aboveas a template. Although it is not intended to limit the presentinvention, for example, a polymerase chain reaction (PCR) is used as thenucleic acid amplification reaction. The enzyme to be used for the PCRis not limited to specific one. A DNA polymerase conventionally used fora PCR can be used. The cDNA obtained according to the method asdescribed above is synthesized from the template RNA with high fidelity.It is desired to conduct the PCR with high fidelity in order toreproduce the sequence from the RNA as accurately as possible. Thus, aDNA polymerase having high fidelity such as an α-type DNA polymerasefrom a thermophilic archaebacterium is preferably used for the cDNAamplification of the present invention. When a heat-resistant DNApolymerase having a 3′-5′ exonuclease activity (e.g., an α-type DNApolymerase from a thermophilic archaebacterium) is used in the cDNAsynthesis step, the same enzyme can be also used in the PCR step.Fidelity of a cDNA synthesis/amplification reaction can be determinedaccording to a modification of the method of J. Cline et al. (J. Clineet al., Nucleic Acids Research, 24:3546-3551 (1996)).

The enzyme may be either an enzyme purified from its original source ora recombinant protein produced by using genetic engineering techniques.The enzyme may be subjected to modification such as substitution,deletion, addition or insertion by genetic engineering techniques orother means. A DNA polymerase having a 3′-5′ exonuclease activityexhibits a proofreading activity which is effective in eliminating abase erroneously incorporated in a synthesized cDNA even if an RNA isused as a template. Thus, it can be used to synthesize a cDNA with highfidelity. In view of cDNA synthesis efficiency, the amount of the enzyme(in terms of polymerase activity) to be used in a reaction volume of 20μl is preferably 1 U or less, more preferably 0.5 U or less. In view ofthe proofreading activity, the amount is preferably 0.01 U or more, morepreferably 0.02 U or more.

By using the method of the present invention, the amount of synthesizedcDNA can be increased, a longer cDNA can be synthesized, and a cDNA canbe synthesized with higher fidelity as compared with the conventionalreverse transcription methods. Furthermore, by combining the method ofthe present invention with related techniques such as cloning techniquesand PCR techniques, it is possible to prepare a cDNA library or toconduct an RT-PCR more efficiently as compared with conventionalmethods, thereby allowing more accurate analysis of an mRNA of which theanalysis by a conventional method was difficult due to the lowexpression level.

The kit for cDNA synthesis of the present invention is a kit to be usedfor the method for synthesizing a cDNA as described above. It is a kitfor synthesizing a cDNA with high fidelity and with high efficiency. Thekit is exemplified by one that contains the enzyme having a reversetranscription activity and the enzyme having a 3′-5′ exonucleaseactivity as described above. The kit may contain a reaction buffer to beused for cDNA synthesis reaction using the enzyme described above,nucleotides and other reagents. cDNA can be synthesized efficiently,with high fidelity and readily by using such a kit.

The kit for amplifying a cDNA of the present invention is a kit to beused for the method for amplifying a cDNA as described above. It is akit for amplifying a cDNA with high fidelity and with high efficiency.The kit is exemplified by one that contains the enzyme having a reversetranscription activity and the enzyme having a 3′-5′ exonucleaseactivity as described above as well as a reagent for conducting a geneamplification reaction. When a PCR is used as a gene amplificationreaction using a cDNA as a template, reagents to be used for theamplification reaction include a heat-resistant DNA polymerase, areaction buffer, nucleotides and other reagents. cDNA can be amplifiedefficiently, with high fidelity and readily by using such a kit. When aheat-resistant DNA polymerase having a 3′-5′ exonuclease activity (e.g.,an α-type DNA polymerase from a thermophilic archaebacterium) is used inthe cDNA synthesis step, the enzyme can be also used in the PCR step asa DNA polymerase for the amplification reaction as described above.

EXAMPLES

The following examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof.

In Examples below, the activities of the respective enzymes areexpressed according to the indications in the instructions attached tothe enzymes.

Example 1 Preparation of RNA

Unless otherwise stated, the RNA used in Examples below was preparedfrom human cultured U-937 cells (ATCC CRL-1593) by using TRIzol reagent(Life Technologies) according to the instructions attached to thereagent. The concentration was then adjusted to 0.66 μg/μl. The purityof the RNA was OD₂₆₀/OD₂₈₀=1.7.

Example 2 Effect of Combination of BcaBEST DNA Polymerase and PyrobestDNA Polymerase

The effect of addition of an enzyme having a 3′-5′ exonuclease activityduring cDNA synthesis was examined using a DNA polymerase derived fromBacillus caldotenax lacking its 5′-3′ exonuclease activity (BcaBEST DNApolymerase, Takara Shuzo) as a reverse transcriptase and a DNApolymerase derived from Pyrococcus sp. (Pyrobest DNA polymerase, TakaraShuzo) as a 3′-5′ exonuclease.

cDNA synthesis reactions were carried out using BcaBEST RNA PCR Kit(Ver. 1.1) (Takara Shuzo). Reaction mixtures each containing theenzyme(s) as described below and an oligo dT primer in a volume of 20 μlwere prepared according to the manual attached to the kit. The reactionmixtures were placed in PCR Thermal Cycler PERSONAL (Takara Shuzo) forreverse transcription reactions at 60° C. for 1, 2, 3 or 4 minutes.After reaction for the predetermined time, they were heated at 98° C.for 5 minutes.

Enzyme 1: BcaBEST DNA polymerase 22 U/reaction system

Enzyme 2: BcaBEST DNA polymerase 42 U/reaction system

Enzyme 3: BcaBEST DNA polymerase 22 U+Pyrobest DNA polymerase 0.017U/reaction system

Enzyme 4: BcaBEST DNA polymerase 42 U+Pyrobest DNA polymerase 0.033U/reaction system

PCRs for amplifying a region of 4.4 kb within the mRNA for transferrinreceptor were carried out using Pyrobest DNA polymerase and 20 μl eachof the reverse transcription reaction mixtures. The PCRs were carriedout according to the manual attached to Pyrobest DNA polymerase asfollows. Reaction mixtures each containing 20 μl of one of theabove-mentioned reverse transcription reaction mixtures, primer 1 foramplifying transferrin receptor (SEQ ID NO:1) and primer 2 foramplifying transferrin receptor (SEQ ID NO:2) in a volume of 100 μl wereprepared. The reaction mixtures were placed in PCR Thermal CyclerPERSONAL and subjected to PCRs (30 cycles of 94° C. for 30 seconds, 65°C. for 30 seconds and 72° C. for 5 minutes).

After the PCRs, 8 μl each of the resulting reaction mixtures wassubjected to electrophoresis on 1% agarose gel using Agarose L03 (TakaraShuzo). The amounts of the PCR products were evaluated by using afluorescence image analyzer FMBIO II Multi-View (Takara Shuzo) tonumerically express the intensity of fluorescence emitted from theagarose gel after electrophoresis and ethidium bromide stainingmeasured. The results for all of the samples converted defining theintensity of fluorescence from the RT-PCR amplification product obtainedfor the combination of the enzyme 1 and the reverse transcriptionreaction time of 3 minutes as 1.00 are shown in Table 1. TABLE 1Reaction time 1 minute 2 minutes 3 minutes 4 minutes Enzyme 1 n.d. n.d.1.00 1.27 Enzyme 2 n.d. 1.03 2.05 2.05 Enzyme 3 n.d. n.d. 1.02 1.53Enzyme 4 n.d. 2.31 2.88 3.19n.d.: not detectable.

When 22 U or 42 U of BcaBEST DNA polymerase was used, the efficiency ofcDNA synthesis was increased by the addition of Pyrobest DNA polymerasewhich has a 3′-5′ exonuclease activity (Table 1, Enzyme 3 or 4). Theincrease in efficiency was particularly remarkable when 42 U of BcaBESTDNA polymerase was used (Table 1, Enzyme 4).

These results show that the efficiency of cDNA synthesis is increased bythe addition of an enzyme having a 3′-5′ exonuclease activity during areverse transcription reaction and that use of a large amount of anenzyme having a reverse transcription activity further increases theefficiency.

As a control experiment, cDNA was synthesized using Titan RT-PCR Kitfrom Boehringer. This kit contains AMV-RTase as an enzyme having areverse transcription activity and Pwo DNA polymerase from Pyrococcuswoesii as an enzyme having a 3′-5′ exonuclease activity.

A cDNA synthesis reaction was carried out at 50° C. for 30 minutesaccording to the manual attached to the kit. After heating at 94° C. for2 minutes according to the manual, a PCR for amplifying a region of 4.4kb within the mRNA for transferrin receptor was carried out using TaqDNA polymerase/Pwo DNA polymerase according to the manual attached tothe kit as follows: 10 cycles of 94° C. for 30 seconds, 55° C. for 30seconds and 68° C. for 4 minutes; 94° C. for 30 seconds, 55° C. for 30seconds and 68° C. for 4 minutes and 5 seconds (the 11th cycle); 94° C.for 30 seconds, 55° C. for 30 seconds and 68° C. for 4 minutes and 10seconds (the 12th cycle); 94° C. for 30 seconds, 55° C. for 30 secondsand 68° C. for 4 minutes and 15 seconds (the 13th cycle); to the 25thcycle while prolonging the time of extension step by 5 seconds percycle.

The RT-PCR product was subjected to electrophoresis on 1% agarose gel.The 4.4-kb amplification product of interest was not detected althoughthe reverse transcription reaction was carried out for 30 minutes.

Example 3 Effect of Combination of BcaBEST DNA Polymerase and Deep VentDNA Polymerase

The effect of the use of Deep Vent DNA polymerase from Pyrococcus sp.GB-D (New England Biolabs) as an enzyme having a 31-5′ exonucleaseactivity was examined.

Experiments were carried out as described in Example 2 using thefollowing enzyme(s).

Enzyme 1: BcaBEST DNA polymerase 42 U/reaction system

Enzyme 2: BcaBEST DNA polymerase 42 U+Deep Vent DNA polymerase 0.033U/reaction system

Enzyme 3: BcaBEST DNA polymerase 42 U+Deep Vent DNA polymerase 0.067U/reaction system

The results are shown in Table 2 (defining the result obtained for thecombination of the enzyme 1 and the reverse transcription reaction timeof 2 minutes as 1.00). TABLE 2 Reaction time 1 minute 2 minutes 3minutes 4 minutes Enzyme 1 n.d. 1.0 2.4 2.4 Enzyme 2 n.d. 1.5 4.7 7.6Enzyme 3 n.d. 2.3 5.0 8.9n.d.: not detectable.

These results show that the efficiency of cDNA synthesis is increased byusing Deep Vent DNA polymerase in place of Pyrobest DNA polymerase as anenzyme having a 3′-5′ exonuclease activity.

Example 4 Effect of Combination of Bst DNA Polymerase, Large Fragmentand Pyrobest DNA Polymerase

The effect of the use of a DNA polymerase derived from Bacillusstearothermophilus lacking its 5′-3′ exonuclease activity (Bst DNApolymerase, Large fragment, New England Biolabs) as an enzyme having areverse transcription activity was examined.

Reverse transcription reactions were conducted as described in Example 2using the following enzyme(s) except that the reverse transcriptionreaction was carried out for 10 minutes.

Enzyme 1: Bst DNA polymerase, Large fragment 8 U/reaction system

Enzyme 2: Bst DNA polymerase, Large fragment 8 U+Pyrobest DNA polymerase0.01 U/reaction system

Enzyme 3: Bst DNA polymerase, Large fragment 8 U+Pyrobest DNA polymerase0.005 U/reaction system

Enzyme 4: Bst DNA polymerase, Large fragment 8 U+Pyrobest DNA polymerase0.0033 U/reaction system

PCRs for amplifying a region of 2.4 kb within the mRNA for transferrinreceptor were carried out. The PCRs were carried out as described inExample 2 except that primer 2 for amplifying transferrin receptor (SEQID NO:2) and primer 3 for amplifying transferrin receptor (SEQ ID NO:3)were used as primers under the following reaction conditions: 30 cyclesof 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 3minutes. The results are shown in Table 3 (defining the result obtainedfor the enzyme 1 as 1.00). TABLE 3 Enzyme 1 1.00 Enzyme 2 1.61 Enzyme 31.67 Enzyme 4 1.92

These results show that the efficiency of cDNA synthesis is increased bythe addition of an enzyme having a 3′-5′ exonuclease activity when BstDNA polymerase, Large fragment was used for a reverse transcriptionreaction as an enzyme having a reverse transcription activity in placeof BcaBEST DNA polymerase.

Example 5 Effect of 3′-5′ Exonuclease Activity on Fidelity During cDNASynthesis

The effect of a 3′-5′ exonuclease activity on the fidelity during cDNAsynthesis was examined using BcaBEST DNA polymerase as an enzyme havinga reverse transcription activity and Pyrobest DNA polymerase as anenzyme having a 3′-5′ exonuclease activity according to a modificationof the method of J. Cline et al. (J. Cline et al., Nucleic AcidsResearch, 24:3546-3551 (1996)).

lacIOZα RNA, which was synthesized using SP6 RNA polymerase (TakaraShuzo) as follows, was used as a template RNA for cDNA synthesis.

lacIOZα is a part of E. coli lactose operon and contains the repressorregion (I), the operator region (O) and the lacZα factor region (Z).

An amplified fragment for the lacIOZα region was obtained by PCR usingE. coli genomic DNA as a template as well as lacIOZα-F primer (SEQ IDNO:4) and lacIOZα-R primer (SEQ ID NO:5). The resulting amplifiedfragment (about 1.9 kb) was cloned into PSCREEN-T™-1 T-Vector(Novagene), a plasmid having SP6 promoter, by TA cloning to obtain aplasmid in which the lacIOZα region was linked downstream from the SP6promoter. An RNA was synthesized from the SP6 promoter region using alinear DNA obtained by digesting the plasmid with a restriction enzymeXhoI (Takara Shuzo) as a template with Competitive RNA Transcription Kit(Takara Shuzo) and SP6 RNA polymerase as follows.

A reaction mixture containing 200 ng of the linear lacIOZα-pSCREEN-T DNAin a volume of 50 μl was prepared according to the manual attached toCompetitive RNA Transcription Kit. The reaction mixture was incubated at37° C. for 2 hours to synthesize an RNA. After reaction, 10 U of DNaseI(Takara Shuzo) was added thereto. The mixture was allowed to stand at37° C. for 1 hour to degrade the DNA. The mixture was then subjected tophenol/chlororform treatment followed by ethanol precipitation to purifythe synthesized RNA. The thus obtained lacIOZα RNA of 1991 bases, whichcontains the SP6 promoter, the repressor region (I), the operator region(O) and the lacZα factor region (Z), was used as a template to carry outthe following experiments.

Reaction mixtures each containing 450 ng of the lacIOZα RNA, 20 pmol ofthe lacIOZα-R primer and the enzyme(s) as described below in a volume of20 μl were prepared according to the manual attached to BcaBEST RNA PCRKit. The reaction mixtures were placed in PCR Thermal Cycler PERSONAL(Takara Shuzo), and incubated at 65° C. for 1 minute and then 30° C. for1 minute. The temperature was raised from 30° C. to 65° C. in 15minutes. The mixtures were then incubated at 65° C. for 15 minutes forreverse transcription reactions, and finally heated at 98° C. for 5minutes.

Enzyme 1: BcaBEST DNA polymerase 42 U/reaction system

Enzyme 2: BcaBEST DNA polymerase 42 U+Pyrobest DNA polymerase 0.033U/reaction system

PCRs for amplifying a 1.9-kb region of lacIOZα were carried out usingPyrobest DNA polymerase and 2.5 μl each of the reverse transcriptionreaction mixtures. The PCRs were carried out according to the manualattached to Pyrobest DNA polymerase as follows. Reaction mixtures eachcontaining 2.5 μl of one of the above-mentioned reverse transcriptionreaction mixtures, lacIOZα-F primer and lacIOZα-R primer in a volume of100 μl were prepared. The reaction mixtures were placed in PCR ThermalCycler PERSONAL and subjected to PCRs (28 cycles of 94° C. for 30seconds, 68° C. for 2 minutes).

After the PCRs, the resulting reaction mixtures were subjected tophenol/chloroform treatment followed by ethanol precipitation to purifythe amplification products. After the amplification products weretreated with a restriction enzyme EcoRI (Takara Shuzo), the wholemixtures were subjected to agarose gel electrophoresis. The bands of theEcoRI-treated fragments were excised after electrophoresis, and theEcoRI-treated fragments were recovered using EasyTrap (Takara Shuzo).Each of the thus obtained EcoRI-treated fragments was ligated with λgt10EcoRI Arms (Takara Shuzo) using Ligation Kit ver.2 (Takara Shuzo). Eachof the ligation mixtures was then subjected to in vitro packaging usingGigapack III gold packaging extract (Stratagene).

100 μl each of serial dilutions of the thus obtained packaging mixtureswas added to 100 μl of a culture of E. coli DH5α (lacZAM15) (OD600=1).The mixtures were allowed to stand at 37° C. for 15 minutes. The wholemixtures were added to 0.7% LB soft agar containing X-gal (1 mg/ml) andIPTG (1.5 mM) (IPTG (+)) or 0.7% LB soft agar containing X-gal but notcontaining IPTG (IPTG (−)), mixed, and overlaid onto LB plates. Theplates were incubated at 37° C. overnight. The number of the formedplaques was counted.

If there is no mutation in lacI within the lacIOZ region,β-galactosidase is not expressed in the absence of IPTG due to theaction of the repressor encoded by the lacI, resulting in white plaques.β-galactosidase is expressed in the presence of IPTG, resulting in blueplaques. On the other hand, if a mutation is introduced into lacI withinthe lacIOZ region, β-galactosidase is expressed even in the absence ofIPTG due to the inactivation of the repressor encoded by the lacI. As aresult, blue plaques are formed on the IPTG (+) and IPTG (−) plates.IPTG (+) IPTG (−) Without mutation blue white With mutation blue blue

Accordingly, blue plaques formed on an IPTG (−) plate indicate mutatedclones. The mutation frequency (mf) can be determined by dividing thenumber of blue plaques formed on an IPTG (−) plate by the number of blueplaques formed on an IPTG (+) plate as shown in the following equation.$\begin{matrix}{{mf} = \frac{{Number}\quad{of}\quad{blue}\quad{plaques}\quad{on}\quad{IPTG}\quad( - )\quad{plate}}{{Number}\quad{of}\quad{blue}\quad{plaques}\quad{on}\quad{IPTG}\quad( + )\quad{plate}}} \\{{mf}\text{:}\quad{mutation}\quad{frequency}}\end{matrix}$

The results are shown in Table 4. TABLE 4 Number of Number of Mutationblue plaques on blue plaques on frequency IPTG (−) plate IPTG (+) plate(mf) Enzyme 1 24 105 0.229 Enzyme 2 27 203 0.133

When the reverse transcription reaction was carried out in the presenceof BcaBEST DNA polymerase alone, the mutation frequency (mf) was 0.229.On the other hand, when the reverse transcription reaction was carriedout in the presence of BcaBEST DNA polymerase and Pyrobest DNApolymerase, the mutation frequency (mf) was 0.133, indicating that thefidelity was about 2-fold higher than that observed in the absence ofPyrobest DNA polymerase.

These results demonstrates that the presence of an enzyme having a 3′-5′exonuclease activity during a reverse transcription reaction increasesnot only the efficiency of cDNA synthesis but also the fidelity.

INDUSTRIAL APPLICABILITY

The present invention is a method that can be used to synthesize a cDNAwith high efficiency of reaction and high fidelity. By combining themethod with related techniques such as cloning techniques and PCRtechniques, it is possible to prepare a cDNA library or to conduct anRT-PCR more efficiently and more accurately as compared withconventional methods, resulting in improvement in mRNA analysis methods.

Sequence Listing Free Text

SEQ ID NO: 1: Designed oligonucleotide primer designated as Primer 1 toamplify transferrin receptor mRNA.

SEQ ID NO: 2: Designed oligonucleotide primer designated as Primer 2 toamplify transferrin receptor mRNA.

SEQ ID NO: 3: Designed oligonucleotide primer designated as Primer 3 toamplify transferrin receptor mRNA.

SEQ ID NO: 4: Designed oligonucleotide primer designated as lacIOZα-F toamplify lacIOZα region of E. coli.

SEQ ID NO: 5: Designed oligonucleotide primer designated as lacIOZα-R toamplify lacIOZα region of E. coli.

1. A method for synthesizing a cDNA, characterized in that the methodcomprises conducting a reverse transcription reaction in the presence ofan enzyme having a reverse transcription activity and an α-type DNApolymerase having a 3′-5′ exonuclease activity.
 2. The method accordingto claim 1, wherein the enzyme having a reverse transcription activityis a heat-resistant reverse transcriptase.
 3. The method according toclaim 2, wherein the enzyme having a reverse transcription activity is aDNA polymerase from a thermophilic bacterium of the genus Bacillus. 4.The method according to claim 3, wherein the enzyme having a reversetranscription activity is a DNA polymerase from Bacillus caldotenax or aDNA polymerase from Bacillus stearothermophilus.
 5. The method accordingto claim 1, wherein the α-type DNA polymerase having a 3′-5′ exonucleaseactivity is an α-type DNA polymerase from a thermophilicarchaebacterium.
 6. A method for amplifying a cDNA, comprisingconducting a gene amplification reaction using a cDNA synthesizedaccording to the method defined by claim 1 as a template.
 7. The methodaccording to claim 6, wherein the gene amplification reaction is a PCR.8. The method according to claim 7, wherein a DNA polymerase for the PCRis the same enzyme as the enzyme having a 31-5′ exonuclease activity. 9.The method according to claim 8, wherein the DNA polymerase for the PCRis an α-type DNA polymerase from an archaebacterium.